The sex determining gene (Sry) on the mouse Y chromosome encodes a protein with two major domains: an amino terminal HMG box and a carboxyl glutamine-rich (Q-rich) domain. The HMG box has been demonstrated to bind to DNA in a sequence-specific manner. The function for the Q-rich domain is unknown. It is primarily encoded by an in-frame insertion of a CAG repetitive sequence. Recently, using the farwestern blotting technique, we have demonstrated that this Sry Q-rich domain is a protein-binding domain and interacts specifically with 3 major proteins (Sips) present in adult testis and embryonic tissues. Further studies demonstrated that the Sips are preferentially expressed in somatic cells of the adult testis and early embryonic tissues and fetal gonads at the time of sex determination. We hypothesize that the Sips play a critical role(s) as co-factors of Sry in testis determination. Three specific aims are proposed in this application to test this hypothesis. First, we will address the question regarding the importance of the HMG box and Q-rich domains in Sry function using transgenic mouse approaches. Chimeric Sry genes will be constructed to harbor various combinations of HMG box and Q-rich domain from different mouse strains that exhibit incompatibility among their Sry genes. They will be evaluated in terms of their ability to mediate testis determination in transgenic XX animals. We plan to determine whether the HMG box or the Q-rich domain is responsible for such incompatibility. Second, interactive cloning techniques, affinity chromatography and protein microsequencing will be used to isolate the cDNAs for the Sips. The interactive properties of their encoded proteins to the Sry Q-rich domain will be confirmed independently. Third, we will characterize the Sip genes in terms of their cDNA sequences, expression patterns, protein structures, interactive domain(s), chromosomal locations, and evolutionary conservation. We will investigate their probable polymorphisms among different musculus and domesticus strains. We hope to relate such polymorphisms to their compatibility in Sry interactions. Their functions are further delineated by gene knockout strategies. Understanding the mechanisms of sex determination in mouse may shed insights on those in humans, and eventually may lead to developments of clinical diagnosis and treatment of gonadal dysgenesis and infertility.